Mechanismen der nicht homologen Endverknüpfung in Zellen der höheren Eukaryonten: Funktionen der DNA Ligase III und Substypen von Histone H1

有证据表明，非同源末端连接（NHEJ）在高等真核生物中是去除DNA双链断裂的核心。我们的研究结果表明NHEJ的两个组件的操作，一个DNA-PK依赖性组件，以快速动力学（D-NHEJ），和DNA-PK-独立的组件（基本或B-NHEJ），以缓慢的动力学和利用尚未确定的因素。当D-NHEJ被抑制时，B-NHEJ而不是同源定向修复（HDR）接管从基因组中去除DSB。有充分的证据表明，DNA连接酶IV参与D-NHEJ。该提议的中心假设是DNA连接酶III参与B-NHEJ。目的是证实这一初步观察结果，并阐明DNA连接酶III在B-NHEJ中的作用。此外，在最近令人兴奋的观察的指导下，我们建议研究H1家族组蛋白在NHEJ中的作用。我们将：1.利用体外质粒试验表征B-NHEJ中DNA连接酶III的功能，并与D-NHEJ中DNA连接酶IV的功能进行比较。2.利用RNA干扰（RNAi）技术敲低DNA连接酶III的表达，研究其在DSB重接和细胞放射杀伤敏感性方面的表现。3.研究组蛋白H1亚型H1.2和H1 X在NHEJ中的作用。作为这些研究的一部分所获得的知识将促进我们对DNA DASB修复机制的理解，也可能是对基因组稳定性的理解。

There is evidence that non homologous end joining (NHEJ) is central to the removal of DNA DSBs in higher eukaryotes. Our results suggest the operation of two components of NHEJ, a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK-independent component (basic or B-NHEJ) that operates with slow kinetics and utilizes as yet unidentified factors. When D-NHEJ is inhibited, B-NHEJ rather than homology directed repair (HDR), takes over to remove DSBs from the genome. There is ample evidence that DNA ligase IV is involved in D-NHEJ. The central hypothesis of this proposal is that DNA ligase III is involved in B-NHEJ. The objective is to confirm this preliminary observation and to elucidate the role of DNA ligase III in B-NHEJ. In additiion, guided by very recent exciting observations we propose to investigate the roles of H1 family histones in NHEJ. We will: 1. Utilize an in vitro plasmid assay to characterize the function of DNA ligase III in B-NHEJ and will compare to that of DNA ligase IV in D-NHEJ. 2. Utilize RNA interference (RNAi) to knock down expression of DNA ligase III and study its manifestations on DSB rejoining and cell radiosensitvivty to killing. 3. Investigate the role of the histone H1 subtypes, H1.2 an H1X, in NHEJ. The knowledge to be acquired as part of these studies will advance our understanding of the mechanismus of DNA DASB repair and probably also of genomic stability.